The LMVR program in Mali is divided into three main units: malaria pathogenesis and immunology, vector studies and Leishmaniasis. Malaria Immunology and Pathogenesis The goals of the malaria pathogenesis/immunology program are: 1) To understand how Malian children with hemoglobin S, alpha-thalassemia, G6PD deficiency, and ABO blood group polymorphisms are protected from Plasmodium falciparum malaria. 2) To develop a profile of the acquisition of malaria immunity in Malian children that incorporates both antibody responses and CD4+ T cell responses to specific malaria antigens. Results will be correlated with blood group polymorphisms. 3) To identify the role of specific PfEMP-1 variants in pathogenesis. We will also study responses to PfEMP1 VAR2CSA in the population since this variant has been associated with placental malaria. 4) To investigate the genetic structure of the malaria parasites in this population and assess how this parasite population changes with time and with selective pressures such as drugs. These goals are being accomplished through a 5-year longitudinal study of 1500 children ranging in age from 6 month to 18 years of age living in 3 villages in Mali (the Kenieroba study). One major effort is to determine the relative protection against malaria conferred by different red blood cell (RBC) polymorphisms, and all the enrolled children have been typed for a series of these polymorphisms. In the past 2.5 years, we have enrolled, diagnosed and treated 2500 episodes of uncomplicated and severe falciparum malaria. Preliminary analysis of our epidemiological data shows that HbS confers significantly greater protection against malaria than other RBC polymorphisms, and that age a surrogate of naturally-acquired immunity is also associated with reduced malaria. We are now investigating the mechanistic basis for this protection by testing the effects of HbS and naturally-acquired IgG on the binding of parasitized RBCs to microvascular endothelial cells (MVECs) a critical event in malaria pathogenesis. Using parasite isolates and freshly obtained RBCs, we have made significant progress in elucidating the mechanism by which G6PD deficiency and iron-deficiency anemia confer malaria protection, and have discovered that HbS and HbC select for particular PfEMP1 variants which we believe contain motifs that might form the basis of a vaccine against severe malaria. We are also investigating the role of uric acid, monokines, and endothelial cell inflammation in malaria pathogenesis. Finally,we are following 300 HbA, HbS, and HbC children weekly for parasitemia and malaria symptoms to explore why HbS is so much more protective against malaria than HbC. To begin to address the acquisition of immunity, we identified a sub-cohort of children, selecting those with sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. These children have been followed for development of humoral and cellular responses to blood-stage antigens of malaria parasites. We have tested the hypothesis that the HbAS children develop accelerated antibody responses to blood-stage malaria antigens as a basis for their protection. However, testing for antibodies to a variety of malaria proteins has shown that HbAS humoral responses are lower than their matched controls. Thus, this cannot be responsible for their increased resistance. Other studies of humoral and cellular immune responses to malaria parasites and specific antigens are also being pursued in these children. A major new initiative this year has been the establishment of in vitro and in vivo tests for parasite drug responses to artesunate and amodiaquine the two antimalarial drugs used in Mali. We are presently monitoring parasite clearance rates of artesunate to begin assessing the artemisinin resistance phenotype in Africa. We have also established a robust in vitro drug susceptibility assay to determine parasite IC50s for these drugs. These responses and parasite genomic data will be followed over time to determine if and how parasite resistance develops in a setting of intense local drug pressure. In the past year, our have supported the submission of 8 abstracts to the 2010 ASTMH meeting, with 4 Malian trainees giving posters or oral presentations. All members of the Fairhurst laboratory, most members of Carole Longs laboratory, and several other trainees in LMVR are presently using data, parasites, or patient blood samples from our Kenieroba protocol. Vectors Malaria vector studies encompass a number of areas of mosquito biology. 1. This year our multi-project study on dry season ecology of mosquitoes in the Sahel (Mali) has addressed one of the most critical and enigmatic issues in vector biology - does An. gambiae persist during the dry season by aestivation(dry season diapauses)? Employing a combination of approaches, we have shown that the major malaria vector An. gambiae can aestivate under natural conditions (Lehman et al., 2010). We have sought to localize the hidden shelters of mosquitoes during the dry season and found that: (i) mosquitoes were markedly clustered in the dry season, (ii) hot spots representing a few houses with high mosquito abundance were found;(iii) mosquito shelters are not inside houses but outdoors. 2. A study on longevity of An. gambiae under field conditions was undertaken to assess reproduction effects on longevity under natural conditions and to develop new means to estimate mosquito fitness(Dao et al,2010). 3. Novel approaches (3D photographic techniques) have been taken to analyzing the structure and dynamics of swarms of male mosquitoes and the consequences of this behavior for reproductive isolation of different An. gambiae molecular forms (Manoukis et al,2009;Diabate et al.,2009). 4. Studies were performed to examine the populations of An. gambiae in another region of Mali with seasonal malaria. It is thought that An. gambiae persist in riverine villages during the dry season and provide the basis for reintroduction of mosquitoes when the rains arrive. Significant evidence for such migration of mosquitoes from the river village to the inland town was reported(Baber et al.,2010). These results suggest strategies for mosquito control in areas with this epidemiology. Sand fly / Leishmaniasis Program The LMVR program on Leishmaniasis and its sandfly vectors is designed to describe the essentially unexamined epidemiology of cutaneous Leishmaniasis in Mali. Based at the MRTC and various field sites, this program has examined the epidemiology of the diseases and the ecology of the sandfly vector. Over three years (2005-2009) the team has measured the prevalence of cutaneous leishmaniasis (CL) and the annual incidence of Leishmania transmission in two study villages in Mali. We found that the chosen villages were endemic for CL with more than 30% of the study population positive to Leishmania (Oliveira et al., 2009). Simultaneously, we identified the vector as the sand fly Phlebotomus duboscqi (Anderson et al.;submitted), and we characterized its most abundant salivary proteins (Kato et al.;2006). Analysis of the human humoral responses specific for the sand fly salivary proteins revealed a strong correlation between anti-saliva antibodies and Leishmania infection (Oliveira et al., unpublished). Most importantly, there is no previous data about cellular immune responses to sand fly salivary proteins in endemic areas. Our preliminary data indicates that these individuals can respond to bites of sand flies in a Th1 or Th2 manner. This may help elucidate why some individuals living in endemic areas present with lesions (likely a Th2 profile), while others are naturally protected (likely a Th1 profile). Moreover, these studies can assist in the discovery of new potential vaccine candidates for Leishmania in saliva.